β-alethine as anti-tumor agent

ABSTRACT

β-alethine is identified as an anti-tumor agent.

GOVERNMENT RIGHTS

This invention was made in the performance of work under grants #HL16,796, #AM 10,628, and #SO7RR-05583-25 with the National Institutes ofHealth, and the United States Government has certain rights therein.

This is a continuation of application Ser. No. 08/316,836, filed Oct. 3,1994, now U.S. Pat. No. 5,643,966 which is a divisional of Ser. No.07/924,977, filed Aug. 5, 1992, now abandoned which is a CIP of Ser. No.07/549,103, filed Jul. 6, 1990 now abandoned.

1. BACKGROUND OF THE INVENTION

The invention provides a method for treating neoplasias with β-alethine.In particular, the invention provides methods for treating a variety ofneoplasias which reduce tumor burden, inhibit tumor growth, and inhibittumor intravascularization, for example from metastasizing tumors.β-alethine has been identified as a compound inducing celldifferentiation and modulating cell growth, phenotypic expression(including bioproduction and function), vitality, and longevity incopending application SN entitled "Beta-Alethine Use in Cell Culture andTherapy"; a correspondence between cell differentiation and reversal ofmalignancy has been suggested; see, for example, "Growth,Differentiation, and the Reversal of Malignancy", Scientific Americanpp. 40-47, January, 1986, and the publications cited therein.

The present application relates to the identification of β-alethine as anon-cell-lineage-dependent anti-tumor compound, and the use ofβ-alethine to induce normalization of the function of a variety ofneoplastic cells, particularly for therapeutic benefits.

2. DISCUSSION OF RELATED ART

β-alethine is known to be produced in vivo as a by-product of metabolicpathways. It is related via these pathways to pantothenic acid, which isa vitamin having known nutritional benefits (see, e.g., J.Reprod.Fert.57: 505-510 (1979), and related compounds have been suggested for use inconjunction with radiotherapy as radioprotectors (J.Med.Chem. 29:2217-2225, 1986; WO 35/00157, Jan. 17, 1985). No other relevant assertedbiological functions of this compound are known to be described in theprior art. The compound is primarily well-known as a starting materialfor the chemical synthesis of related compounds (see, e.g., Japanesepatent applications (83) 198461; (83) 46063A2; (81) 156256A2; (81)104861A2; (80) 124755; (75) 62932; (80) 07222; and U.S. Pat. Nos.2,835,704 and 4,552,765; for examples of the preparation of β-alethine,β-aletheine, and pantetheine, and derivatives and intermediates of thesecompounds, used in the synthesis of coenzyme A and its analogues).

3. SUMMARY

The invention accordingly provides methods for the recognition,normalization, and elimination of neoplastic cells particularly for thetreatment of cancer. Within the scope of the present invention,β-aletheine, the reduced form of β-alethine, is to be considered thebiological equivalent of β-alethine for purposes of practicing theinvention, as β-alethine is readily reduced to β-aletheine in vivo, forexample by abundant intracellular thiol compounds, such as glutathionein mammals, including humans. Both compounds have the advantage ofhaving inherent antioxidative properties; however, β-alethine ischemically more resistant to autoxidation than β-aletheine, and the useof β-alethine in the present invention is generally preferred for thisreason.

4. BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1-3 illustrate early and late tumor development in mice inoculatedwith non-secreting myeloma cells (NS-1) and treated with varying dosesof β-alethine.;

FIG. 4 is a three-dimensional composite of the data of FIGS. 1-3;

FIG. 5 is a 85 degree clockwise rotation of the illustration of FIG. 4;and

FIG. 6 illustrates modulation of tumor in two mice with differingamounts of β-alethine.

FIGS. 7, 8, and 9 illustrate stimulation of antibody production by humanand mice cells exposed to β-alethine.

5. DETAILED DESCRIPTION OF THE INVENTION

As indicated above, β-alethine is a known compound [(H₂ NCH₂ CH₂ (C═O)NHCH₂ CH₂ S)₂ and Formula I following], commonly produced by oxidationof the corresponding monosulfide, β-aletheine [H₂ NCH₂ CH₂ (C═O)NHCH₂CH₂ SH and Formula II following], which is unstable in air and aqueoussolutions (The Merck Index, 9th edition (#221), Merck & Co., Rahway,N.J., USA): ##STR1## Both compounds are stabilized as their acid salts,particularly their hydrogen halide salts, and especially theirhydrochloride salts. Various techniques for the synthesis of β-alethinebased on deblocking of (N,N'-bis-carbobenzoxy)-blocked β-alethine aredescribed in the literature (carbobenzoxy is often abbreviated as CBZ);however, most of the known procedures result in unsatisfactory yield orpurity of product, or both. Accordingly, it is preferred that β-alethinefor use in the processes of the invention be prepared by processes whichensure purity of product and preferably also maximize yield, for exampleby the process of the invention comprising coupling N-CBZ-blockedβ-alanine to N-hydroxysuccinimide to produce the corresponding activeester, which is then coupled to cystamine prepared by oxidation ofcysteamine with hydrogen peroxide; the product, CBZ-blocked β-alethine,is then recovered and deblocked. The process is described in detail inthe Examples, and provides a high-yield, high-purity product suitablefor pharmaceutical use.

According to the invention, β-alethine appears to regulate a set ofgeneric differentiation mechanisms that are not cell-lineage specificand that are common to cells regardless of phenotypic specialization.Consistent with this premise, the use of β-alethine according to theinvention as an anti-tumor agent is not significantly dosage-dependentwith respect to cell lineage, phenotype, or point of intervention in thecell cycle, except as noted below. For in vivo applications, from about10 pg of β-alethine/kg of body weight is recommended, particularlyamounts from about 10 pg/kg up to about 200 μg/kg, and moreparticularly, up to about 100 μg/kg, which may be administered by anycustomary route including parenterally (such as i.p.) or anally inconjunction with conventional carriers such as physiological saline fornon-oral routes, or orally with appropriate entero-coating. Thecompounds are preferably administered on a daily or alternate-dayregimen as described more fully below, until the desired results areachieved, although other regimens, such as weekly or biweekly regimensmay suffice, particularly when results are apparent; decreases indosages as normalization progresses or tumor burden is reduced may besuitable, and may be preferable to avoid too-rapid regression of tumorsaccompanied by excessive physiological stress on the organism. Use ofamounts of β-alethine substantially in excess of those required toobtain differentiation, normalization of cell function, decrease oftumor burden, or other results noted herein are not recommended, asexcessive dosages may be counterproductive or at least ineffective. Forin vitro applications for normalization of neoplastic cells, dosagesstarting from about 10 pg/ml culture are suggested, with daily orbi-daily replenishment.

It is contemplated that β-alethine is useful for the treatment ofneoplasias of cells of living organisms in general, including mammalian,especially human, reptilian, avian, and plant cells, owing to thecommonality of results obtained with corresponding dosages observed inexperiments reported herein and unreported experiments. According to theinvention, β-alethine comprises an anti-tumor compound which normalizescell function (i.e., increases insufficient function or decreases excessfunction). β-alethine specifically functions to (1) inhibit tumorgrowth, especially that of malignant tumors; (2) regress tumors,especially malignant tumors; (3) inhibit tumor metastasis; and (4)normalize growth characteristics of neoplastic cells; and/or (5) improverecognition and/or elimination of neoplastic cells.

In cancer applications, neoplastic cells or immunocytes, or bothneoplastic cells and immunocytes, are exposed to β-alethine to promotedifferentiation of the cells and normalize the cell cycle. Treatment oftumor cells is effectively segregated from treatment of the immunocytesby removing immunocytes from the afflicted mammal, including humans. Theimmunocytes are then treated in culture with β-alethine, or with acombination of β-alethine and tumor cells derived from the afflictedmammal, until either the immunocytes are activated, or the tumor cellsare completely attenuated for health reasons, respectively. Theactivated immunocytes preferably devoid of metastatic tumor cells arethen reinjected into the mammal. β-alethine is useful in vivo forreducing soft (hematolymphoid) tumor burden, particularly in mammals,especially in humans, and inhibiting intravascularization of tumorcells, especially cells of metastasizing tumors. The compounds are thususeful for reducing tumor burden, by inhibiting tumor growth or byinhibiting tumor metastasis, or both. In particular, β-alethine iscontemplated to be useful in the treatment of numerous soft and lymphoidmalignant tumors, such as lymphomas; leukemias; hepatocellular tumors;liver tumors; and Hodgkin's disease; especially tumors such as myelomas.β-alethine is contemplated inter alia as useful in the treatment ofneoplasia 1) prophylactically; 2) as a primary therapy for inhibitingtumor growth, particularly that of slowly-growing tumors; and 3) as asupplemental therapy pursuant to surgical intervention for removal ordebulking of tumors, particularly virulent or primary tumors. Treatmentwith β-alethine has been found to regress tumors, reduce tumor mass,inhibit tumor growth, inhibit tumor metastasis, and inhibit tumorascites production.

It is recommended that anti-tumor therapy commence at the earliest tumorstage possible, particularly to avoid peripheral physiologicalcomplications caused by the presence or metastasis of large tumors.β-alethine for tumor therapy is administered by any convenient route asnoted above, for example i.v. or i.p., in a suitable conventionalcarrier such as physiological saline at at least therapeutic thresholdamounts; from about 1 ng/kg body weight up to about 100 μg/kg areparticularly suitable, depending upon the stage of the tumor. Dosagestoward the higher. end of the therapeutic range are recommended forStage II tumors and above, whereas dosages toward the lower end of therange are suitable for Stage I or incipient tumors. For cancerprophylaxis, dosages ranging from about 10 pg/kg body weight, preferablyfrom about 1 ng/kg up to about 100 μg/kg body weight are contemplated.Therapeutic regimens of alternate days for the dosages noted above forcancer treatment are suitable, and appear to be preferable, based on theobservation that in vivo induction of biochemicals such as enzymesthought to be responsive to β-alethine therapy appear to follow chemicalstimulation by about 48 hrs. Prophylactic regimens may be followed on adaily basis.

At least at the dosage levels indicated, β-alethine appears to be asubstantially non-toxic compound in healthy mice, with no observedadverse side-effect.

For in vitro applications, immunocytes and optionally tumor cellsremoved by methods described in the prior art are treated by exposingthe cells to an appropriate amount of β-alethine prior to reinjection ofthe immunocytes into the afflicted mammal according to methods describedin the prior art. Techniques for removing and maintaining cells inculture (immunocytes in particular), treating these cells withimmunopotentiating agents in cell culture, and reinjecting theseimmunopotentiated cells into mammals are described in the prior art, forexample, Immune Responses to Metastases (volumes I and II, 1987, CRCPress, Boca Raton, Fla., USA), Ann. Surg. (201: 158-163, 1985), NewEngland J. Med. (319: 1676-1680, 1988), Cancer Immunol. Immunother. (26:1-10, 1988), Surgery (pp. 262-272, August 1986), and Current Protocolsin Immunology (volumes 1 and 2, Green Publishing Associates andWiley-Interscience, New York, N.Y., USA) incorporated herein byreference. Broadly suitable techniques for removing cells from theafflicted mammal include needle or core biopsies or surgical removal oforgans, tumors, or humoral fluids. Outgrowth from needle biopsies, ortreatment of core biopsies or tissues with collagenase or sievingthrough appropriately sized sterile meshes to disrupt organization ofsolid tissues, comprise exemplary methods for establishing cultures ofcells, in vitro. According to the present invention, similarly removedcells are treated with β-alethine for an appropriate amount of time incell culture, and similarly reinjected into the afflicted mammal, theimprovement in technique being the treatment of the cells withβ-alethine. In certain instances, it may be desirable to removeβ-alethine prior to reinjection of the immunocytes into the afflictedmammal by washing the cells as described in the prior art. For in vitroapplications according to the invention, exposure of the cells to fromabout 10 pg β-alethine included per ml of cell culture media (based on adensity of from about 10⁵ to about 10⁷ cells/ml) for an appropriateamount of time, for example from about 30 minutes to four weeks or untilthe desired immunopotention is evident as described in the prior art, issuggested. In particular, in vitro stimulation of antibody production byhuman peripheral blood leukocytes or murine splenocytes is mostefficacious between 10 pg and 1 μg β-alethine/ml cell culture afterabout four to six days of exposure (see, e.g, Examples V and VI). Theculture should be replenished with the compound as necessary, generallyon a daily basis; treatment on an alternate-day or biweekly basis maysuffice, depending upon the desired results. During this period,exposure of the cells to immunogenic substances or cells other thanextracted tumor cells, such as allogeneic serum proteins in the cellculture media, should be minimized to preclude diversion of theimmunological response to non-tumor targets.

6. EXAMPLES

I. Preparation of β-alethine:

β-alethine was produced by deblocking N,N'-bis-carbobenzoxy (CBZ)blocked β-alethine produced as follows:

A. Preparation of N,N'-bis-(CBZ)-β-alethine or5,5-Bis-[N-carbobenzoxy-β-alanyl)-2-aminoethyl] disulfide.

A solution of dicyclohexylcarbodiimide (23.3 g) was added to a solutionof N-CBZ-β-alanine (24.84 g) and N-hydroxy-succinimide (12.92 g) in atotal volume of about 500 ml of dry 10% acetonitrile in dichloromethane.Dicyclohexylurea (24.51 g) precipitated as a by-product upon formationof the active ester. The active ester was dried to an oil and trituratedwith anhydrous ethyl ether. The precipitate was resuspended indichloromethane and additional dicyclohexylurea was allowed toprecipitate. The resulting dichloromethane solution of active ester wasfiltered and added to a previously prepared solution of cystamine (8.5g). The desired product, N,N'-bis-(CBZ)-β-alethine precipitated fromthis mixture. The mother liquor, anhydrous ether, dichloromethaneextracts of the product, and the anhydrous ether extract of the activeester recovered above were dried and recombined to augment the yield ofproduct. The product was substantially insoluble in water, hot (aboveabout 70° C.) ethyl acetate, and hot (above about 30° C.) ether, andthese can be used to further extract impurities. The product can also berecrystallized from dimethyl sulfoxide with acetonitrile or water, andagain rinsed with ethyl acetate and ether. The latter process results ina 1° C. increase in product melting point, from 180 to 181° C.(uncorrected). Yields of N,N'-bis-(CBZ)-β-alethine of up to theoreticalyields are contemplated; yields of 85-90% of theory have been routinelyobtained. When dried over P₂ OS₅, in vacuo, the product appears toretain one mole equivalent of water, and was analyzed accordingly as themonohydrate.

Anal. Calcd. for C₂₆ H₃₄ N₄ O₆ S₂.H₂ O: C, 53.78; H, 6.25; N, 9.65.

Found: C, 54.23; H, 6.56; N, 9.66. Sample analyzed by Ruby Ju,Department of Chemistry, University of New Mexico, Albuquerque, N.M.

B. Deblocking of CBZ-blocked β-alethine obtained from I.A., above[preparation of β-alethine.2HCl; or N,N'-bis-(β-alanyl)-cystamine; orN,N'-bis-(β-alanyl-2-aminoethyl) disulfide].

Complete removal of the carbobenzoxy group was accomplished according toprocedures described in J.Am.Chem.Soc. 86: 1202-1206 (1964),incorporated herein by reference. After deblocking with four equivalentsof hydrogen bromide in glacial acetic acid per mole of theN,N'-bis-(CBZ)-β-alethine for 14 hours, the β-alethine was purified byprecipitation with acetonitrile, rinsing with anhydrous ethyl ether,resuspension in water and filtering, and precipitation of the mixed saltproduct with acetonitrile. Initial yields were in excess of 80% oftheoretical maximum yields. β-alethine was converted to thehydrochloride salt by passing the preparation over a 30 ml×15 cm longcolumn of Dowex AG 1×8 (chloride form) (Dow Chemical Corp., Midland,Mich., USA) which had been previously prepared by eluting with 1M KCland rinsing thoroughly with DI (deionized) water. Neutralization withCa(OH)₂ and recrystallization of the β-alethine HCl from water withacetonitrile resulted in fine needles which melted at 224-225° C.(uncorrected).

Anal. Calcd. for Calcd for C₁₀ H₂₂ N₄ O₂ S₂.2HCl: C, 32.69; H, 6.59; N,15.25.

Found: C, 32.52; H, 6.69; N, 15.32. Sample analyzed by Ruby Ju, Dept. ofChemistry, University of New Mexico, Albuquerque, N.M.

C. Characterization of β-alethine: [¹³ C]-NMR; [¹ H]-NMR; and IR spectraof β-alethine.

    __________________________________________________________________________          a   b    c      d     e                                                    S--CH.sub.2 CH.sub.2 --N H--N--C═O O--C--CH.sub.2 CH.sub.2 --N f         ═ ═════════════.                                     dbd.═══════.d                                     bd.═══════.db                                     d.>                                                                             #STR2##                                    [.sup.13 C]-NMR                                                               β-alethine                                                                     37.59                                                                             39.04                                                                              172.79 32.9  36.71                                                                              --                                           [.sup.1 H]-NMR                                                                β-alethine                                                                     2.524                                                                             3.094       2.694 3.367                                                                              --                                             ═ ═════════════.                                     dbd.═══════.d                                     bd.═══════.db                                     d.>  bis-(CBZ)-  β-alethine                                                #STR3##                                    (DMSO)                                                                              2.740                                                                             3.309                                                                              8.085  2.254 3.192                                                                              7.24                                            a b c d e f                                                                IR (cm.sup.-1)                                                                      a   b    c      d     e    f                                              ═ ═════════════.                                     dbd.═══════.d                                     bd.═══════.db                                     d.>                                                                             #STR4##                                    β-alethine                                                                     660w      3250w --         3270v                                             1555w-s  2970s-w                                                           1286m 1462s                                                                   1620s  1620s                                                                   1128s                                                                        ═ ═════════════.                                     dbd.═══════.d                                     bd.═══════.db                                     d.>  bis-(CBZ)-  β-alethine                                                #STR5##                                    --             3345s  --         3345s                                            1545m  1535s                                                                  1640s  1270m                                                                     1682s                                                                    a b c d e f                                                                 __________________________________________________________________________                       (R is a benzyl moiety in this table.)                  

β-alethine is unusual in that changes in pH [neutralization with Ca(OH)₂] cause pronounced shifts in the positions and intensities of IR bands.

Peaks (HCl salt): 3270s, 3170s, 2970s, 2700w, 2550w, 2020w, 1657s,1595m, 1560s, 1450s, 1409m, 1390w, 1354w, 1325m, 1300w,shoulder/1252m/shoulder, 1188m, 1129m, 1097m, 1079w, 1030w, 950w, 905w,829m.

Peaks (neutralized): 3250w, 3180w, 2940m/broad, 2375s, 2230s, 2157s,1936w, 1620s, 1555w, 1462s, 1432 shoulder, 1400m, 1342m, 1286m, 1217m,1188m, 1128s, 1020m, 810w, 719m, 660w.

Bis-(CBZ)-β-alethine displays few of the resonances present inβ-alethine.

Peaks: 3345s, 3310s, 1682s, 1640s, 1545m shoulder, 1535s, 1450w, 1427w,1375w, 1332m, 1270m, 1231m, 1178w, 1120w, 1030m/broad.

II. Inoculation of Mice with NS-1 Myeloma Cells.

NS-1 myeloma cells (ATCC TIB 18, P3/NS1/1-Ag4-1) were employed asinoculant; these cells have proven to be about 90% effective inestablishing myelomas in mice according to the exemplified procedure,and the untreated myelomas are substantially fatal within about twoweeks.

The cells were grown for several passages (preferably one week) in asterile environment consisting of RPMI 1640 (Whittaker M. A.Bioproducts, Walkersville, Md., USA) containing 10% fetal calf serum(Hyclone Laboratories, Logan, Utah, USA), 2 mM L-glutamine, 5,000 unitsof penicillin, and 5 mg streptomycin in 75 cm² polystyrenetissue-culture flasks (Corning Glassworks, Corning, N.Y., USA) in ahumidified chamber at 37° C. and under 6% CO₂. To assure NS-1propagation in vivo it is essential to remove DMSO (the cryostatic agentdimethyl sulfoxide) through several medium changes and dilutions; thisalso serves to maintain the cells in log-phase growth. Female BALBc/Jmice were injected i.p. with 10⁴ cells in 0.1 ml of standardphosphate-buffered saline as soon as possible after weaning, transport,and indexing, as it has been found that the NS-1 cell line employed doesnot generally perform optimally in animals which are mature or whichhave equilibrated with their environment. The mice were maintained withWayne Rodent Blox (Wayne Research Animal Diets, Chicago, Ill., USA) adlib. and tap water.

III. Treatment of Inoculated Mice (Example II) with β-alethine (earlyintervention).

A. Concentrations of β-alethine as obtained above (Example I) of 1ng/kg, 1 pg/kg, 10 μg/kg, and 100 μg/kg (based on the body weight of theinoculated mice) were injected i.p. in 0.1 ml physiological salinestarting the second day after tumor inoculation (day 2), and continuingevery Monday, Wednesday, and Friday through day 47. This regimen waspredicated on the observation that enzymes thought responsive to thesecompounds and which may play a role in the reported results are induced48 hours after chemical stimulation. The inoculated mice were comparedto a) untreated controls and b) to carrier-injected (saline-injected)controls.

B. Conclusions

β-alethine is effective for preventing the onset of NS-1 myeloma inBALBc/J mice over the concentration range from 10 pg β-alethine/kg mouseto 100 μg/kg mouse. Without treatment, 75% of the mice in the experimenteither had to be euthanized or died as the result of tumor development.At doses of β-alethine below the effective threshold [i.e., below about10 pg/kg, or at about 10 pg/kg or 1 ng/kg (data not illustrated for thelatter)] one-third to two-thirds of the animals ultimately contractedtumor. At dosages approaching the maximal effective dose (i.e., aboveabout 10 μg/kg, or at about 10 μg/kg or 100 μg/kg), only one mousedeveloped a palpable tumor, which persisted for 20 days but eventuallyregressed. FIGS. 1-3 illustrate early and late tumor development (basedon weight of mouse not attributable to normal weight gain) in micetreated with decreasing concentrations of β-alethine (10 μg, 10 μg, and10 pg per kg mouse, respectively). In FIG. 3, the biphasic curve in thecenter illustrates early and late tumor development in these mice andcorresponds to two deaths at this dosage (10 pg/kg mouse) of β-alethinewhich is the therapeutic minimal threshold for this compound in thismodel (FIG. 6). Normal weight gain of the mice is slightly inhibited at100 μg/kg (FIG. 1), but not significantly at 10 μg/kg (FIG. 2). At 10μg/kg (FIG. 2) one mouse developed a palpable tumor which persisted for20 days but eventually regressed. At effective dosages of theantineoplastic compound β-alethine (from about 10 pg/kg body weight toabout 10 μg/kg body weight), there is a striking difference between theweights of the mice (reflecting tumor burden) in the untreated controlgroup (the vehicle-injected control group), compared with the mice inthe treatment group.

The effectiveness of β-alethine in the treatment of NS-1 myeloma isfurther illustrated in FIG. 4, comprising a three-dimensionalrepresentation of the study; and in FIG. 5, comprising a 85° clockwiserotation of the illustration of FIG. 4, with higher doses of the drug inthe front to no doses of the drug in the back. The controls inoculatedwith tumor (far left, FIG. 4) gain tumor and therefore weight (Z-axis)at an accelerated rate with respect to the day of the study (Y-axis).Mice receiving high doses of β-alethine (far right) develop at anear-normal rate and show no signs of chronic tumor. Ridges or increasesin elevation illustrate tumor development at the lower concentrations(farther left on the X-axis) which are coded for the morphology of thedeveloping tumor (Ta=ascites and Ts=solid tumors) and for deathsresulting from either ascites or solid tumors (Da or Ds respectively).Complete regression is indicated by R at the point at which the tumor isno longer palpable. When normalized to the initial weights of the micein the control group and plotted (analyzed as in FIGS. 1-5), controlmice (physiological saline injections only) displayed normal growth anddevelopment approximating the growth and development of the micereceiving tumor and 10 μg β-alethine/kg mouse (FIGS. 2 and 4). This isfurther illustrated in FIG. 6 in a mouse in which a tumor is modulatedwith different concentrations of β-alethine (below). The only departurefrom the normal growth curve coincides with the appearance anddisappearance of a palpable tumor.

A tumor appeared and regressed without any signs of malaise in a mouseundergoing a therapy of 10 μg/kg β-alethine (FIG. 2). Tumors developingin untreated controls with some exceptions typically promoted ascitesdevelopment, while tumors developing in mice undergoing therapy withβ-alethine were with few exceptions solid masses; since it thus appearsthat β-alethine promotes consolidation of tumor into discrete masses,β-alethine is projected to be valuable in the design of treatmentregimens involving surgical debulking.

IV. Treatment of Inoculated Mice (Example II) with β-alethine (lateintervention).

A single mouse developing a tumor late in the experiment (at 4 on theX-axis, FIG. 4, approximately 40 days after the 10 pg β-alethine/kgmouse treatment was discontinued) was treated with 100 μg β-alethine/kgmouse to determine the effect of late therapeutic intervention on thetreatment of the myeloma (FIG. 6). Massive log-phase growth of tumorpersisted along the right side of the mouse from shoulder to hip and inthe abdomen for 10 to 14 days after treatment with the higher dose wasbegun, indicating pronounced infiltration of the tumor intoextra-peritoneal tissues, and a considerable lag phase before thetreatment became effective. The growth then ceased, and was followed byrapid reduction of both the tumor mass and the weight of the mouse.There was a brief period in which the malaise subsided and the mouse'srough coat improved. This coincided with a temporary stabilization(approximately 1 week) of the mouse's weight suggesting that the tumoralso stabilized during this time. This was followed by anotherprecipitous drop in the mouse's weight and obvious decrease in the tumormasses. The mouse was euthanized when the weight returned to normal eventhough a palpable mass remained in the abdomen. At the time the mousewas euthanized, phlebitis was evident in the extremities, possiblyresulting from the processing of the tumor equivalent of roughly 10% ofthe body weight per day. After correcting for necrosis evidenthistologically, it was estimated that between 85 and 90% of the originaltumor was either necrotic or resorbed at the time of euthanasia.Considering the rate of resorption, there would have been completeregression of the tumor if the therapy had been maintained for the fullmonth.

Wasting of the tumor and not the mouse proper was confirmed by weighingthe debulked carcass. In this mouse, unlike untreated controls, therewas no gross evidence of infiltration of organs by the tumor, and theremaining tumor appeared necrotic (yellowish-green and granular like anold sponge). Histological examination of the tissues indicated remainingtumor cells in the skeletal muscle adjacent to the abdominal wall, thesubcutaneous tissue, and a mammary gland. Hepatic, urogenital, andgastrointestinal tumors, as well as a variety of other tumors, have beenconsistently observed in untreated mice, indicating the highly invasiveand metastatic nature of the NS-1 cell line; however, in the treatedmouse, none of the remaining organs contained tumor cells. An apparentpathological bone fracture was observed in this mouse, but no tumorcells were evident in the marrow of this bone. It was thus tentativelydetermined that the bone demineralized due to rapid growth of the tumor,a process requiring calcium and phosphate, and that subsequent rapidextraskeletal deposition of calcium phosphate as the tumor was resorbedresulted in the fracture.

Based on this and other studies, it is recommended that in the presenceof aggressive neoplasia (e.g., stage II or above tumors), at least theprimary tumor should be debulked surgically in conjunction withβ-alethine therapy, preferably before or very soon after β-alethinetherapy is commenced. It is further recommended that in some instances,β-alethine initial dosages of about 100 μg/kg used in late interventiontherapy be gradually reduced to slow the resorptive process and permitthe organism to adjust to the therapy. Gradual decreases of dosages (onan alternating 48 hour regimen) from about 100 μg/kg down to about 1ng/kg are suggested as the tumor responds to the therapy.

V. Differentiation of a Peripheral Lymphoid Organ with β-alethine.

FIG. 7 illustrates data from a series of experiments designed to assessthe effect of β-alethine on non-antigen-specific immunoglobulinsynthesis and secretion in vitro by human peripheral blood leukocytes(HPBLs), generally characterized as a peripheral lymphoid organprimarily populated by medium-sized, mature lymphocytes. In theseexperiments, blood was taken from healthy male humans; the blood wasthen defibrinated on glass beads, and the leukocytes separated bycentrifugation (buffy coat technique). Residual red blood cells werelysed with a brief treatment with 0.85% ammonium chloride. Theleukocytes were counted and dispensed into 24 well tissue culture trays(LUX, Flow General, McClean, Md., USA) in 1 ml of RPMI 1640 basal mediumsupplemented with penicillin, streptomycin, 10% fetal calf serum, andL-glutamine. β-Alethine was added to test cultures at variousconcentrations in 30 microliter doses. The cells were harvested asindicated in FIG. 7 at various times between 72 and 144 hours of cultureand tested for antibody production using a conventional protein-Afacilitated plaque assay (A-PFC or Ig-PFC). In this assay, protein-A wascovalently conjugated to washed sheep red blood cells (SRBC's) usingchromium chloride in saline (6 mg/100 ml) and used as target in theplaque assay. In addition, aliquots of cells were also tested forproliferation status by treating them with 0.5 μCi of tritiatedthymidine (6-9 Ci/mole) followed by assessing the level of incorporationof radioactivity into newly synthesized DNA. FIG. 7 shows thatβ-alethine stimulated the HPBLs to produce immunoglobulin in adose-dependent manner at approximately 60 times the untreated controllevels. The optimal concentration was about 5 nanograms/ml culture. Theproliferation index indicated a low level of increased thymidineincorporation at 5 ng/ml doses. This 2-to 3-fold increase overbackground has minor significance as compared to truly proliferativestimulants such as LPS (lipopolysaccharide) or PHA (phytohemagglutinin),which under similar conditions result in the incorporation of about200,000 cpm of radioactivity, approximately 100 to 200-fold that ofcontrol levels. It was concluded that the level of proliferationobserved in the experiment was attributable to differentiation-dependentproliferation rather than to independent proliferative processesstimulated by β-alethine, or to an increase in both the survival ofcells and the retentive capacity for deoxyribonucleic acids associatedwith viable cells.

VI. Differentiation of a Central Lymphoid Organ with β-alethine.

FIG. 8 shows data from a series of experiments designed to study theeffect of β-alethine on murine splenocyte production of non-specificimmunoglobulin. The assays and culture techniques were the same as thosedescribed above (Example V) for the HPBL model. The animals, 4 to 6 weekold female BALBc/J mice, were sacrificed by cervical dislocation, andtheir spleens aseptically removed and pressed through 90 mesh stainlesssteel screens. After several washes with the complete medium the cellswere counted and dispensed into the 24 well trays and test cultures weredosed with the β-alethine. These cultures were also harvested overvarious culture times of from 72 to 144 hours of culture, and tested forantibody production and proliferation, as shown in FIG. 8. FIG. 8illustrates that β-alethine markedly stimulated the murine splenocytesto produce immunoglobulin in a dose-dependent manner, with anillustrated optimum at approximately 10 ng/ml dosages. In this casethere was no significant stimulation of proliferation, based on thethymidine assay described in Example V.

VII. Differentiation of a Central Lymphoid Organ with β-alethine.

FIG. 9 shows data from a series of experiments designed to study theeffect of β-alethine on murine splenocyte production of specific typesof immunoglobulin. The assays and culture techniques were the same asthose described in Examples V and VI for the murine splenocyte model,with the exception that rabbit antibodies which are specific for thegamma chain of mouse antibodies is used to detect murine splenocytesproducing IgG. The plaque assay is described in detail in the prior artby N. K. Jerne, et. al., in Transplant. Rev. 18: 130-191 (1974)incorporated herein by reference. The animals, 4 to 6 week old femaleBALBc/J mice, were sacrificed by cervical dislocation, and their spleensaseptically removed and pressed through 90 mesh stainless steel screens.After several washes with the complete medium the cells were counted anddispensed into the 24 well trays (LUX, Flow General, McClean, Md., USA)in 1 ml of RPMI 1640 basal medium supplemented with penicillin,streptomycin, 10% fetal calf serum, and L-glutamine. β-alethine wasadded to test cultures at various concentrations in 30 microliter doses.The cells were harvested as indicated in FIG. 9 at various times between72 and 144 hours of culture and tested for antibody production using aconventional protein-A facilitated plaque assay (A-PFC or Ig-PFC). Inthis assay, protein-A was covalently conjugated to washed sheep redblood cells (SRBC's) using chromium chloride in saline (6 mg/100 ml),and the conjugate was used as target in the plaque assay. FIG. 9illustrates that β-alethine markedly stimulates the murine splenocytesto produce IgG in a dose-dependent manner, with an illustrated optimumat approximately 10 ng/ml dosages and about four days into the study.

What is claimed is:
 1. A method of treating a non-hematolymphoidneoplasia sensitive to β-alethine in a mammal in need of such treatmentcomprising administering to said mammal an amount of β-alethine, orphysiologically compatible salt thereof, sufficient of effect saidtreatment.
 2. The method according to claim 1 wherein said neoplasia isa metastatic tumor.
 3. The method according to claim 1 wherein saidadministered β-alethine inhibits tumor growth.
 4. The method accordingto claim 1 wherein said β-alethine inhibits tumor metastasis.
 5. Themethod according to claim 1 wherein β-alethine is administeredintravenously.
 6. The method according to claim 1 wherein β-alethine isadministered intraperitoneally.
 7. The method according to claim 1wherein said amount is from about 1 ng/kg body weight to about 100 μg/kgbody weight.
 8. The method according to claim 1 wherein said neoplasiais a liver tumor, a mammary tumor, a urogenital tumor, agastrointestinal tumor or a muscle tumor.
 9. The method according toclaim 8 wherein said neoplasia is a liver tumor.
 10. The methodaccording to claim 8 wherein said neoplasia is a mammary tumor.
 11. Themethod according to claim 8 wherein said neoplasia is a urogenitaltumor.
 12. The method according to claim 8 wherein said neoplasia is agastrointestinal tumor.
 13. The method according to claim 8 wherein saidneoplasia is a muscle tumor.
 14. A method of treating anon-hematolymphoid neoplasia sensitive to β-alethine in a mammal in needof such treatment comprising:i) removing immunocytes from said mammaland placing said immunocytes in a culture medium, ii) exposing saidimmunocytes to β-alethine, or a physiologically compatible salt thereof,and iii) reintroducing immunocytes resulting from step (ii) into saidmammal,wherein, in step (ii), said immunocytes are exposed to β-alethinefor a period of time and in an amount such that, upon saidreintroduction, said immunocytes recognize and eliminate cells of saidneoplasia, and wherein, in step (iii), said immunocytes are reintroducedin an amount sufficient to effect said treatment.
 15. The method ofclaim 14, wherein the mammal is human.
 16. The method of claim 14,wherein the immunocytes are additionally exposed to tumor cellsextracted from the afflicted mammal prior to reinjection.
 17. The methodaccording to claim 14 wherein said amount of β-alethine is at least 10pg per ml of said culture medium.
 18. The method according to claim 14wherein said amount of β-alethine is in the range of 10 pg/ml culturemedium to 1 μg/ml culture medium.
 19. The method according to claim 14wherein said immunocytes are exposed to β-alethine for from 30 minutesto four weeks.